| Candida infection can cause many infectious diseases,threatening human life and health.Fungi multiple resistance and its biofilm formation result in the decrease of its sensitivity to antifungal drugs.How to improve the effect of antifungal drugs has become one of the hot spot of current research.Ketoconazole(KCZ)is now mainly used to treat skin fungal infection via percutaneous delivery and all kinds of deep fungus disease althoughits severe hepatotoxicity limites its clinical application.To reduce the side effect of KCZ,combination way can be selected to achieve synergistic antifungal effect.Curcumin(CUR)is a natural polyphenolic compound used as a spice and food coloring agent in Asia.Many research works have showed that CUR's cooperation with azole antifungal agents reduces the minimum inhibition concentration(MIC)value in vitro treatment of fungi,showing synergistic effect.However,low water solubility and poor bioavailability of CUR and KCZ limit their clinical application.The polymer micelle drug system can improve the drug solubility in water,enhance physical and chemical properties,prolong the biologic half life of drug in the blood and control release rate of drug.Polymer micelle drug system plays an important role in the clinical research and application of traditional antifungal drugs with potentially far-reaching consequences.In this study,methoxy poly(ethylene glycol)-b-poly(?-caprolactone)(MPEG-PCL)was synthesized via ring-opening polymerization of MPEG and ?-caprolactone with ethyl ether-hydrochloride as catalyst.Structure of MPEG-PCL was confirmed by 1H-NMR and FT-IR,and its molecular weight was measured by GPC.The drug-loaded micelles were prepared through thin membrane hydration method.Optimization of preparation technology was accomplished by evaluating the drug-loading amounts and the entrapment efficiency of KCZ.When the ratio of MPEG/PCL was 1:1.7,the weight of polymer was 70 mg,hydration temperature and hydration time were 60 min and 30?,respectively,the EE and DL of KCZ-M reach its maximum,85.27±1.75 % and 11.31±1.05 %,respectively.Under the same condition,the EE and DL of CUR-M were 91.19±0.83 % and11.90±0.11 %,respectively.The drug-loaded micelles endowed the two drugs' slow controlled release with water-solubility enhanced to 85 and 82900 folds higher than the corresponding raw drugs,respectively.Transmission electric microscopy(TEM)and dynamitic light scattering(DLS)characterization revealed the formation of ketoconazole-and curcumin-loaded micelles with an average size of 44.70±1.91 nm and 39.56±0.19 nm,respectively.Fourier infrared spectroscopy(FT-IR)and X-ray diffraction(XRD)analysis showed that KCZ-M and CUR-M was successful fabricated with amorphous form of the crude drugs in micelles.Dynamic dialysis in vitro release experiments illustrated that the drug-loaded micelles could control drug release,with a slow release behavior.The kinetic release model of KCZ-M in vitro was abided by first order kinetics meaning thatdrug release rate is proportional to the drug concentration.CUR-M accorded with zero order kinetics signifying that drug release quantity is proportional to the release time.In vitro antifungal activity test,chequerboard test and inhibition zone test indicated that efficacy of ketoconazole-loaded micelles was improved by introduction of curcumin-loaded micelles at low dosage(1.40~22.5 ?g/m L)with a low fractional inhibitory concentration index(FICI=0.073).Biofilm formation inhibition assay also demonstrated that participation of curcumin-loaded micelles obviously strengthened the inhibition of fungal biofilms formation induced by ketoconazole-loaded micelles using XTT reduction mothod and laser scanning confocal microscope.The study suggested that the high synergistic activity of combinations is encouraging and the MPEG-PCL micelle is a potential drug delivery system for the combination of ketoconazole and curcumin.This study provided a new way for other antifungal drugs,showingits potential clinical application value and development prospect. |
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